Lab 4: Macromolecules
The four major kinds of macromolecules are carbohydrates, lipids, proteins and
nucleic acids. Carbohydrates, proteins and nucleic acids are polymers made of smaller
subunits called monomers. Carbohydrates are polysaccharides made of monosaccharide
monomers. Proteins are polypeptides made of amino acids held together through peptide
bonds and nucleic acids are polynucleotides made of nucleotide subunits. Lipids are not
considered polymers; they have non-repetitive components. A fat for instance is made
from one molecule glycerol plus three fatty acids. A phospholipid is built with glycerol,
two fatty acids and a phosphate group. Scientists use chemical assays to test for the
presence of a certain molecule or a class of molecules. Often the test (or the assay) will
indicate with a change of color of a particular substance is present. If no reaction is
visible, the test result is negative.
Test for protein (Biuret test)
Place on dropper of egg albumin (known protein source) in a test tube. Add one dropper
of concentrated NaOH /CuSO4 solution (Biuret solution). What color is produced? This
color indicates the presence of protein in the egg albumin. If smaller polypeptides were
present, the color would be pink or very pale purple. Biuret detects peptide bonds. If you
want to test an unknown substance, mix equal amounts of your protein substance with
Biuret reagent.
Test for reducing sugars (Benedict’s solution)
Add 1 ml glucose solution (known sugar source) to 2-3 ml of Benedict’s solution in a test
tube. Label your tube and place it in boiling water for 5 minutes. Observe the color
change during that time. This test is an oxidation-reduction reaction in which the sugar
becomes oxidized as copper (in the Benedict’s solution) is reduced. A positive reaction
may give colors from khaki to yellow to orange to red to brown, depending on the
concentration of the sugar. To test an unknown substance for sugar content, mix the
substance with 1-2 times the amount of Benedict’s solution.
Test for Fat (Sudan III dye)
Add a small amount of Sudan dye to about 2 ml of water in a test tube. The dye will not
dissolve. Then add 1-2 ml of vegetable oil (known fat source) to another test tube. Add
Sudan dye to it and shake. The Sudan dye will dissolve in the fat. If you combine the
contents of the two tubes, you will observe that the dye will only go in solution in the oil
phase, not in the aqueous phase.
Test for Starch (Iodine solution)
Put 1 ml of starch solution (known starch source) in a test tube. Add one or two drops of
iodine solution. What happens? Does the same color change occur if glucose instead of
starch is added to iodine? To test an unknown substance, add a few drops of iodine
solution to the substance and mix.
Test for Calcium ions (Ca2+) (Oxalic Acid)
© B. Woelker, 2008
Mix 20-30 drops of 0.1 M CaCl2 (calcium chloride -known calcium source) solution with
a few drops of O.1M oxalic acid (C2H2O4). What happens? To test an unknown
substance, add 4-8 drops of oxalic acid are added to the substance and mixed.
Test for Chloride Ions (Cl-) (Silver Nitrate)
Mix 20-30 drops of 0.1 M CaCl2 solution with 2-4 drops of 0.1 M AgNO3 (silver nitrate).
What do you observe?
BACKGROUND INFORMATION
Benedict Test: Reducing vs. Non-Reducing Sugars:
•
Reduction is defined as the gain of electrons and oxidation is defined as the loss of
electrons. Obviously, these two processes go hand-in-hand! For every substance that
gains an electron, another substance has to lose one (Conservation of Matter Law)!
•
Reducing sugars have free aldehyde or ketone groups. The carbonyl group (C=O)
is common to both aldehyde and ketone groups. The double bond serves as a source
of the electrons that reduces other substances/molecules (the sugar itself will lose the
electrons and be oxidized). Most monosaccharides and some disaccharides are
reducing sugars since they can exist for brief periods of time in a structural (chain)
form. The polysaccharides are non-reducing sugars since their basic units
(monomers) always exist in a closed-ring form.
Aldehyde: R-C=O
Ketone: R-C=O
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H
R
Glucose (aldose): ring formation. If the sugar molecule can be in solution in the linear
and the ring form, it still has the aldehyde group and is a reducing sugar.
Sucrose (table sugar) is a disaccharide and consists glucose and fructose
© B. Woelker, 2008
In sucrose the aldehyde on C1 of glucose or the ketone on C2 of fructose are not
present any longer since the 1-2 glycosidic bond in sucrose does not allow the glucose
nor the fructose to flip back into their linear forms. Sucrose therefore is a nonreducing sugar.
•
The chemical test for the presence of a reducing sugar is Benedict’s solution - a blue
solution of copper sulfate (Cu2+SO4=). In the presence of a reducing sugar the
following reduction reaction occurs:
•
Cu 2+ + 2e–
•
The Cu atoms are unstable and therefore react with oxygen to form cupric oxide
(Cu2O), which is a brick red precipitate. This is a general test for the presence of
aldose or ketose sugars but it cannot distinguish between these sugars (for example
you couldn’t distinguish glucose from fructose).
Cu˚
Starch:
•
Starch is a complex polysaccharide and is primarily a storage product in plants. It is a
polymer of glucose (basic repeating unit). It is made up of amylose (400-500 glucose
units with alpha 1-4 bonds) and amylopectin (approximately 1,000 glucose units with
alpha 1-4 and 1-6 bonds). Side branching produces a non-rigid molecule in
comparison to cellulose with its beta 1-4 bonds (leads to a rigid linear polymer).
•
Starch can be hydrolyzed (covalent bonds broken through the addition of water
molecules) to glucose with hydrochloric acid (HCl) and to maltose with salivary
(alpha) amylase (also known as ptyalin).
•
The simple, specific test for the presence of starch is iodine. Iodine is a starch
indicator since you obtain a positive color change to blue-black.
© B. Woelker, 2008
Biuret Test
Biuret is a condensation compound of urea, equivalent to two molecules of urea less one
of ammonia. It is a white solid soluble in hot water and decomposes at 186–189 °C. The
parent compound can be prepared by heating urea above the melting point at which
temperature ammonia is expelled.
2 CO(NH2)2
 H2N-CO-NH-CO-NH2 + NH3
↑
The term biuret can also apply to the functional group and class of organic compounds
with the general structure RHN-CO-NR-CO-NHR where R is an organic residue.
The biuret reagent is used in the biuret protein assay, a chemical test for proteins not
because the reagent contains biuret but because both biuret and proteins have the same
response to copper. However, the biuret test will also return a positive result in contact
with polypeptides as it detects the peptide bonds.
The Biuret Reagent includes sodium hydroxide and copper sulfate. The blue reagent turns
violet in the presence of proteins, and changes to pink when combined with short-chain
polypeptides.
© B. Woelker, 2008